The present invention relates to the regulatory response of plants to environmental stresses such as cold and to drought. More specifically, the present invention relates to genes which regulate the response of a plant to environmental stresses such as cold or drought and their use to enhance the stress tolerance of recombinant plants into which these genes are introduced.
Environmental factors serve as cues to trigger a number of specific changes in plant growth and development. One such factor is low temperature. Prominent examples of cold-regulated processes include cold acclimation, the increase in freezing tolerance that occurs in response to low non-freezing temperatures (Guy, C. L., Annu. Rev. Plant Physiol. Plant Mol. Biol. 41:187-223 (1990)); vernalization, the shortening of time to flowering induced by low temperature (Lang, A., in Encyclopedia of Plant Physiology, Vol.15-1, ed. Ruhland, W. (Springer, Berlin), pp. 1489-1536 (1965)); and stratification, the breaking of seed dormancy by low temperature (Berry, J. A. and J. K. Raison, in Encyclopedia of Plant Physiology, Vol. 12A, eds. Lange, O. L., Nobel, P. S., Osmond, C. B. and Ziegler, H. (Springer, Berlin), pp. 277-338 (1981)). Due to the fundamental nature and agronomic importance of these processes, there is interest in understanding how plants sense and respond to low temperature. One approach being taken is to determine the signal transduction pathways and regulatory mechanisms involved in cold-regulated gene expression.
Strong evidence exists for calcium having a role in low temperature signal transduction and regulation of at least some COR (cold-regulated) genes. Dhindsa and colleagues (Monroy, A. F., et al, Plant Physiol. 102:1227-1235 (1993); Monroy, A. F., and R. S., The Plant Cell, 7:321-331 (1995)) have shown that, in alfalfa, calcium chelators and calcium channel blockers prevent low temperature induction of COR genes and that calcium ionophores and calcium channel agonists induce expression of COR genes at normal growth temperatures. Similarly, Knight et al (The Plant Cell 8:489-503 (1996)) have shown that cold-induced expression of the Arabidopsis thaliana COR gene KIN1 is inhibited by calcium chelators and calcium channel blockers. These results suggest that low temperature triggers an influx of extracellular calcium that activates a signal transduction pathway that induces the expression of COR genes. Consistent with this notion is the finding that low temperature evokes transient increases in cytosolic calcium levels in plants (Knight, M. R. et al, Nature 352:524-526 (1991); Knight, H., et al., The Plant Cell 8:489-503 (1996)). In addition, low temperatures have been shown to stimulate the activity of mechanosensitive calcium-selective cation channels in plants (Ding, J. P. and B. G. Pickard, Plant J. 3:713-720 (1993)).
Recent efforts have led to the identification of a cis-acting cold-regulatory element in plants, the C-repeat/DRE (Yamaguchi-Shinozaki, et al., The Plant Cell 6:251-264 (1994); Baker, S. S., et al., Plant. Mol. Biol. 24:701-713 (1994); Jiang, C., et al., Plant Mol. Biol. 30:679-684 (1996)). The element, which has a 5 base pair core sequence for CCGAC, is present once to multiple times in all plant cold-regulated promoters that have been described to date; these include the promoters of the COR15a (Baker, S. S., et al, Plant. Mol. Biol. 24:701-713 (1994)), COR78/RD29A (Horvath, D. P., et al, Plant Physiol. 103:1047-1053 (1993); Yamaguchi-Shinozaki, K., et al., The Plant Cell 6:251-264 (1994)), COR6.6 (Wang, H., et al., Plant Mol. Biol. 28:605-617 (1995)) and KIN1 (Wang, H., et al, Plant Mol. Biol. 28:605-617 (1995)) genes of Arabidopsis and the BN115 gene of Brassica napus (White, T. C., et al, Plant Physiol. 106:917-928 (1994)). Deletion analysis of the Arabidopsis COR15a gene suggested that the CCGAC sequence, designated the C-repeat, might be part of a cis-acting cold-regulatory element (Baker, S. S., et al., Plant Mol. Biol. 24:701-713 (1994)). That this was the case was first demonstrated by Yamaguchi-Shinozaki and Shinozaki (Yamaguchi-Shinozaki, K., et al., The Plant Cell 6:251-264 (1994)) who showed that two of the C-repeat sequences present in the promoter of COR78/RD29A induced cold-regulated gene expression when fused to a reporter gene. It was also found that these two elements stimulate transcription in response to dehydration and high salinity and thus, was designated the DRE (dehydration, low temperature and high salt regulatory element). Recent studies by Jiang et al (Jiang, C., et al., Plant Mol. Biol. 30:679-684 (1996)) indicate that the C-repeats (referred to as low temperature response elements) present in the promoter of the B. napus BN115 gene also impart cold-regulated gene expression.
U.S. Pat. Nos. 5,296,462 and 5,356,816 to Thomashow describe the genes encoding the proteins involved in cold adaptation in Arabidopsis thaliana. In particular the DNA encoding the COR15 proteins is described. These proteins are significant in promoting cold tolerance in plants.
A need exists for the identification of genes which regulate the expression of cold tolerance genes and drought tolerance genes. A further need exists for DNA constructs useful for introducing these regulatory genes into a plant in order to cause the plant to begin expressing or enhance their expression of native or non-native cold tolerance genes and drought tolerance genes. These and other needs are provided by the present invention.
DNA in isolated form is provided which includes a sequence encoding a binding protein capable of selectively binding to a DNA regulatory sequence which regulates expression of one or more environmental stress tolerance genes in a plant. The binding protein is preferably capable of regulating expression of one or more environmental stress tolerance genes in a plant by selectively binding to a DNA regulatory sequence which regulates the one or more environmental stress tolerance genes. In one embodiment, the binding protein is a non-naturally occurring protein formed by combining an amino acid sequence capable of binding to a CCG regulatory sequence, preferably a CCGAC regulatory sequence with an amino acid sequence which forms a transcription activation region which regulates expression of one or more environmental stress tolerance genes in a plant by regulating expression of one or more environmental stress tolerance genes when the binding protein binds to the regulatory region.
DNA in isolated form is also provided which includes a promoter and the sequence encoding the binding protein. In one variation, the promoter causes expression of the binding protein in a manner which is different than how the binding protein is expressed in its native state. For example, the promoter may increase the level at which the binding protein is expressed, express the binding protein without being induced by an environmental stress and/or express the binding protein in response to a different form or degree of environmental stress than would otherwise be needed to induce expression of the binding protein. The promoter may also be inducible by an exogenous agent. The promoter can also be selected with regard to the type or types of plant tissues that the binding protein will be expressed as well as when in the plant""s life the promoter will function to regulate expression of the binding protein.
A nucleic acid construct capable of transforming a plant is also provided which includes a sequence encoding a binding protein capable of selectively binding to a DNA regulatory sequence which regulates expression of one or more environmental stress tolerance genes in a plant. The binding protein is preferably capable of regulating expression of one or more environmental stress tolerance genes in a plant by selectively binding to a DNA regulatory sequence which regulates the one or more environmental stress tolerance genes. The nucleic acid construct may be an RNA or DNA construct. Examples of types of constructs include, but are not limited to DNA and RNA viral vectors and plasmids.
A nucleic acid construct capable of transforming a plant is also provided which includes a sequence which when transformed into a plant expresses a binding protein capable of selectively binding to a DNA regulatory sequence which regulates one or more environmental stress tolerance genes in the plant. The binding protein preferably regulates expression of one or more environmental stress tolerance genes in the plant by selectively binding to a DNA regulatory sequence which regulates the one or more environmental stress tolerance genes.
In one variation of the above constructs, the construct also includes a promoter which regulates expression of the binding protein encoding sequence. The promoter may optionally be homologous or heterologous relative to the binding protein encoding sequence. The promoter and binding protein encoding sequence may also optionally be native to the same or a different plant species. In one variation, the promoter causes expression of the binding protein in a manner which is different than how the binding protein is expressed in its native state. For example, the promoter may increase the level at which the binding protein is expressed, express the binding protein without being induced by an environmental stress and/or express the binding protein in response to a different form or degree of environmental stress than would otherwise be needed to induce expression of the binding protein. The promoter may also be inducible by an exogenous agent. The promoter can also be selected with regard to the type or types of plant tissues that the binding protein will be expressed as well as when in the plant""s life the promoter will function to regulate expression of the binding protein.
A binding protein in isolated form is also provided which is capable of selectively binding to a DNA regulatory sequence which regulates expression of one or more environmental stress tolerance genes in a plant. The binding protein is preferably capable of regulating expression of one or more environmental stress tolerance genes in the plant by selectively binding to a DNA regulatory sequence which regulates the one or more environmental stress tolerance genes.
A recombinant binding protein expressed within a plant is also provided which is capable of selectively binding to a DNA regulatory sequence in the plant which regulates expression of one or more environmental stress tolerance genes in the plant. The recombinant binding protein is preferably capable of regulating expression of one or more environmental stress tolerance genes in the plant by selectively binding to a DNA regulatory sequence which regulates the one or more environmental stress tolerance genes. The recombinant binding protein may be native or non-native to the plant. Further, the recombinant binding protein may be homologous or heterologous relative to the DNA binding protein present in the plant in which the binding protein is expressed.
A transformed cell of an organism is also provided which includes a recombinant sequence encoding a binding protein capable of selectively binding to a DNA regulatory sequence which regulates expression of one or more environmental stress tolerance genes in a plant. The binding protein is preferably capable of regulating expression of one or more environmental stress tolerance genes in a plant by selectively binding to a DNA regulatory sequence which regulates the one or more environmental stress tolerance genes. The transformed cell may be a unicellular organism such as a bacterium, yeast or virus, or from a multicellular organism such as a fungus or a plant.
A transformed cell is also provided which includes a promoter and a sequence encoding a binding protein where at least one of the promoter and sequence under regulatory control of the promoter is recombinant. Optionally, one or both of the promoter and sequence under regulatory control of the promoter is not native to the cell. In one variation, the promoter causes expression of the binding protein in a manner which is different than how the binding protein is expressed in its native state. For example, the promoter may increase the level at which the binding protein is expressed, express the binding protein without being induced by an environmental stress and/or express the binding protein in response to a different form or degree of environmental stress than would otherwise be needed to induce expression of the binding protein. The promoter may also be inducible by an exogenous agent. The promoter can also be selected with regard to the type or types of plant tissues that the binding protein will be expressed as well as when in the plant""s life the promoter will function to regulate expression of the binding protein.
A transformed cell is also provided which includes a recombinant binding protein expressed within the cell which is capable of selectively binding to a DNA regulatory sequence in the plant which regulates expression of one or more environmental stress tolerance genes in the plant. The binding protein is preferably capable of regulating expression of one or more environmental stress tolerance genes in the plant by selectively binding to a DNA regulatory sequence which regulates the one or more environmental stress tolerance genes. The binding protein may be native or non-native to the cell.
A transformed plant with modified environmental stress tolerance gene expression is also provided. In one embodiment, the transformed plant includes one or more environmental stress tolerance genes; a DNA regulatory sequence which regulates expression of the one or more environmental stress tolerance genes; and a recombinant sequence encoding a binding protein capable of selectively binding to the DNA regulatory sequence.
In another embodiment, the transformed plant includes one or more environmental stress tolerance genes; a DNA regulatory sequence which regulates expression of the one or more environmental stress tolerance genes; a sequence encoding a binding protein capable of selectively binding to the DNA regulatory sequence; and a recombinant promoter which regulates expression of the sequence encoding the binding protein.
In yet another embodiment, the transformed plant includes one or more environmental stress tolerance genes; a recombinant DNA regulatory sequence which regulates expression of the one or more environmental stress tolerance genes; and a sequence encoding a binding protein capable of selectively binding to the DNA regulatory sequence.
In yet another embodiment, the transformed plant includes at least one recombinant environmental stress tolerance gene; a DNA regulatory sequence which regulates expression of the at least one environmental stress tolerance gene; and a sequence encoding a binding protein capable of selectively binding to the DNA regulatory sequence.
In yet another embodiment, the transformed plant includes at least one recombinant environmental stress tolerance gene; a DNA regulatory sequence which regulates expression of the at least one environmental stress tolerance gene; and a recombinant binding protein expressed by the plant which is capable of selectively binding to the DNA regulatory sequence.
A method for altering an environmental stress tolerance of a plant is also provided. In one embodiment, the method includes transforming a plant with at least one copy of a gene encoding a binding protein capable of binding to a DNA regulatory sequence which regulates one or more environmental stress tolerance genes in the plant; expressing the binding protein encoded by the gene; and stimulating expression of at least one environmental stress tolerance gene through binding of the binding protein to the DNA regulatory sequence.
In another embodiment, the method includes transforming a plant with a promoter which regulates expression of at least one copy of a gene encoding a binding protein capable of binding to a DNA regulatory sequence which regulates one or more environmental stress tolerance genes in the plant; expressing the binding protein encoded by the gene; and stimulating expression of at least one environmental stress tolerance gene through binding of the binding protein to the DNA regulatory sequence.
In another embodiment, the method includes transforming a plant with one or more environmental stress tolerance genes whose expression is regulated by a DNA regulatory sequence; and expressing a binding protein capable of binding to the DNA regulatory sequence and activating expression of the one or more environmental stress tolerance genes.
According to any one of the above embodiments of the present invention, the binding protein may optionally be selected such that it selectively binds to a member of a class of DNA regulatory sequences which includes the subsequence CCG or more particularly one of the following subsequences: CCGM, CCGAT, CCGAC, CCGAG, CCGTA, CCGTT, CCGTC, CCGTG, CCGCA, CCGCT, CCGCG, CCGCC, CCGGA, CCGGT, CCGGC, CCGGG, AACCG, ATCCG, ACCCG, AGCCG, TACCG, TTCCG, TCCCG, TGCCG, CACCG CCCG, GACCG, GTCCG, GCCCG, GGCCG, ACCGA, ACCGT, ACCGC, ACCGG, TCCGA, TCCGT, TCCGC, TCCGG, CCCGA, CCCGT, CCCGC, CCCGG, GCCGA, GCCGT, GCCGC, and GCCGG. The binding protein may also be selected such that the binding protein includes an AP2 domain.
In each of the above embodiments, the level of expression of the binding protein may be the same or different than the level of expression of the binding protein in its native state. Expression of the binding protein in the transformed cell may be regulated by a recombinant promoter which may have the effect of increasing the level at which the binding protein is expressed, expressing the binding protein without being induced by an environmental stress and/or expressing the binding protein in response to a different form or degree of environmental stress than is otherwise needed to induce expression of the binding protein. Expression may also be induced by an exogenous agent. Expression may also be limited to selected types of plant tissues or selected periods in the plant""s life based on which promoter is used. By selecting in what tissues and when in a plant""s life the binding protein is expressed, in combination with the selecting how the binding protein is expressed (level of expression and/or type of environmental or chemical induction), an incredible range of control over the environmental stress responses of a plant can be achieved by the present invention.
In each of the above embodiments, the binding protein comprises an amino acid sequence which is capable of binding to a DNA regulatory sequence which regulates one or more environmental stress tolerance genes. In a preferred embodiment, the binding protein further comprises a transcription activation region which acts in concert with the binding sequence to regulate expression of one or more environmental stress tolerance genes in the plant by regulating expression of one or more environmental stress tolerance genes. The environmental stress tolerance gene, DNA regulatory sequence, sequence encoding the binding sequence, and the sequence encoding the transcription activation region may each independently be native or non-native to the plant and may each independently be homologous or heterologous relative to each other.
Optionally, the binding protein satisfies one or more of the following requirements:
the binding protein comprises an AP2 domain which comprises a consensus sequence sufficiently homologous to any one of the consensus sequences shown in FIGS. 19A, 19B, or 19C that the binding protein is capable of binding to a CCG regulatory sequence, preferably a CCGAC regulatory sequence;
the binding protein comprises an AP2 domain which comprises a consensus sequence shown in FIGS. 19A, 19B or 19C;
the binding protein comprises an AP2 domain which comprises the amino acid residues shown in FIGS. 19D or 19E;
the binding protein comprises an AP2 domain which is sufficiently homologous to at least one of the AP2 domains shown in the application such that it is capable of binding to a CCG regulatory sequence, preferably a CCGAC regulatory sequence;
the binding protein comprises one of the AP2 domain sequences shown in this application, including, but not limited to SEQ. I.D. Nos. 2, 13, 15, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65,67, 69, 71, 73,75, 77, 79,81, 83, 85, 87, 89, 91, 93, and 95;
the binding protein comprises a sequence which comprises one of the amino terminus domains shown in FIG. 20 (it is noted that the sequence need not be at the amino terminus of the binding protein);
the binding protein comprises the consensus sequence for the amino terminus domains shown in FIG. 20, (it is noted that the sequence need not be at the amino terminus of the binding protein);
the binding protein comprises a sequence which comprises one of the carboxy terminus domains shown in FIG. 21A (it is noted that the sequence need not be at the carboxy terminus of the binding protein);
the binding protein comprises the consensus sequence for the carboxy terminus domains shown in FIG. 21A (it is noted that the sequence need not be at the carboxy terminus of the binding protein); and
the binding protein comprises the consensus sequence for the carboxy terminus domains shown in FIG. 21B (it is noted that the sequence need not be at the carboxy terminus of the binding protein).
The amino acid sequence encoding the binding protein may be a naturally occurring sequence such as the ones shown in SEQ. ID. Nos. 2, 13, 15, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, and 95 or may be a non-naturally occurring sequence. It is noted, however, that binding proteins according to the present invention are intended to encompass non-naturally occurring sequences which are derivatives of the classes of binding proteins taught herein. For example, additional binding proteins may be constructed using one of the AP2 domains taught herein or the consensus sequence of these AP2 domains. It may be desirable to include with the AP2 domain a transcription activation region. The transcription activation region may be native to the plant or non-native to the plant in which the binding protein will be used. For example, the sequence may include a subsequence which encodes a binding domain for the DNA regulatory sequence fused to a transcription activating region, such as the transcription activating region of VP16 or GAL4. Optionally, one can include in the binding protein one of the amino terminus domains, the consensus sequence for the amino terminus domain, one of the carboxy terminus domains and/or the consensus sequence for the carboxy terminus domains. It is noted that the amino terminus domain may be positioned away from the amino terminus of the new binding protein and the carboxy terminus domain may be positioned away from the carboxy terminus of the new binding protein.
Optionally, the binding protein can be viewed as comprising one of the amino terminus domains, the consensus sequence for the amino terminus domain, one of the carboxy terminus domains and/or the consensus sequence for the carboxy terminus domains. It is noted that the amino terminus domain may be positioned away from the amino terminus of the new binding protein and the carboxy terminus domain may be positioned away from the carboxy terminus of the new binding protein.
A method is also provided for identifying from a cDNA library of at least a portion of a plant genome a gene sequence encoding a protein capable of binding to a target DNA regulatory sequence. In one embodiment, the method comprises
taking a microorganism which includes a target DNA regulatory sequence for one or more environmental stress tolerance genes, a transcription activator for activating expression of a reporter gene, and a reporter gene whose expression is activated by a protein which includes a binding domain capable of binding to the target DNA regulatory sequence and an activation domain capable of activating the transcription activator;
fusing sequences from a cDNA library of at least a portion of a plant genome to a sequence which encodes a functional activation domain in the microorganism;
introducing the fused sequences into the microorganism; and
selecting microorganisms which express the reporter gene, expression of the reporter gene indicating expression of a fusion protein which includes a binding domain for the target DNA regulatory sequence and the activation domain; and
identifying the gene sequence from the cDNA library introduced into the microorganism. The target DNA regulatory sequence may optionally include the subsequence CCG or the subsequence CCGAC. This embodiment of the invention also relates to DNA in substantially isolated form, nucleic add constructs capable of transforming a plant, cells, and transformed plants which include a gene sequence identified by this method.
While the present invention is described with regard to the use of binding proteins which can bind to a DNA regulatory sequence that regulates environmental stress tolerance genes in a plant, it is noted that these same binding proteins can also be used to regulate genes other than environmental stress tolerance genes by placing these other genes under the regulatory control of the DNA regulatory sequence. For example, protein kinases that induce cold and drought inducible genes can be regulated by placing a protein kinase gene under the control of a promoter whose expression is regulated by the DNA regulatory sequence. PCT/US97/23019 (Intl Publication Number WO 98/26045) describes protein kinases that when constitutively expressed, induce cold and drought inducible genes. The ATCDPK1a and the ATCDPK1 constitutive protein kinase coding regions (PCT/US97/23019) can be isolated by PCR and inserted into the drought and cold inducible promoters described in Example 8 by one skilled in the art. The expression of these ATCDPK1 constitutive protein kinase coding regions (PCT/US97/23019) from the drought and cold inducible promoters will increase the drought and cold tolerance of plants and should be synergistic with the the drought and cold tolerance induced by CBF expression under inducible promoters.